Project Summary Our objective is to develop 6K-F17 as an antimicrobial agent that can be prescribed with tobramycin for the treatment of chronic, antibiotic-resistant Pseudomonas aeruginosa infection in cystic fibrosis (CF) patients. CF affects over 70,000 people worldwide1. Even with the current array of antibiotics, 80-95% of CF patients will ultimately succumb to respiratory failure brought on by chronic bacterial infections3. As a result, there is a significant unmet need to develop new antimicrobials for chronic, antibiotic-resistant infections in CF patients. CF is a genetic disease arising from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that encodes a chloride ion transporter4. Impaired trans-epithelial chloride transport leads to dehydrated airway secretions and a lack of airway mucus clearance5, 6. As a result, individuals with CF are prone to chronic, biofilm-based bacterial infections, bronchiectasis and respiratory failure6, 7In particular, chronic infection with P. aeruginosa has been shown to lead to more rapid lung function decline, a lower quality of life, and premature death8-11. Chronic infection with P. aeruginosa is particularly challenging to treat as this strain continually adapts to environmental challenges, including the formation of a mucus-like biofilms. These biofilms protect bacteria from natural host defense mechanisms and make them more resistant to small molecule antibiotics 5, 12-18. Progress toward developing treatments for biofilm infections has been made through the use of cationic antimicrobial peptides (CAPs). CAPs are found naturally in a wide variety of organisms ranging from plants to humans and constitute a major component of the innate immune system19-21. Importantly, these natural CAPs kill bacteria by destroying their membranes ? in effect ?blowing up? the bacteria by lodging and accumulating in their membranes - rather than by targeting a specific bacterial protein or biochemical pathway. This mechanism impedes the advent of bacterial resistance to CAPs. Recently, our team developed a novel peptide, called 6K-F17, that can disrupt biofilms created by P. aeruginosa and kill the bacteria using a novel mechanism of membrane association. Due to its unique mechanism of action, 6K-F17 synergizes with antibiotics, such as tobramycin, to kill antibiotic-resistant strains of P. aeruginosa. Building from this work, the objective of this proposal is to generate the optimal dose, pharmacokinetics and toxicology profiles for 6K-F17 in mice. The specific aims are to: 1) produce sufficient amounts of 6K-F17 and standardize analytical and potency assays; 2) determine the optimal effective dose of 6K-F17 to eliminate P. aeruginosa infection in mice; and 3) establish a preliminary acute toxicology profile for 6K-F17 in normal mice. Successful completion of these studies will advance an exciting antimicrobial agent for the treatment of antibiotic-resistant P. aeruginosa, and potentially other antibiotic-resistant lung infections, in CF patients.